Concentrating immunochemical test strip

ABSTRACT

A method and device for determining the presence of an analyte in a sample suspected of containing the analyte is disclosed. The method involves contacting a test solution containing the sample and a first member of a specific binding pair with an end portion of a strip of bibulous material capable of being traversed by the test solution through capillary action. The first member of a specific binding pair is capable of binding the analyte. The strip contains a second member of a specific binding pair integral therewith for concentrating and non-diffusively binding the first sbp member at a small situs on the strip separated from the end portion of the strip. The detectible signal is produced in relation to the presence of the analyte in the test solution. The test solution passes through the situs as the test solution traverses the bibulous material. After the test solution has been allowed to traverse at least a portion of the strip, the strip is contacted with a developer solution containing members of a signal producing system in a manner that provides contact of the developer solution with the small situs following its contact with the test solution. The strip is then contacted with any remaining members of the signal producing system. The detectible signal produced at the situs is then compared with the signal detectible at a portion of the strip other than the situs to determine the analyte in the sample. In one embodiment of the invention the signal produced at the small situs has a sharp-edged distinctive pattern that provides a sharp contrast to the signal produced at adjacent sites on the strip when analyte is present in the test solution.

This is a continuation of pending application Ser. No. 701,464, filed2/14/85, incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The ability to employ naturally occurring receptors or antibodiesdirected to specific compounds in assaying for the presence of acompound of interest has created a burgeoning immunoassay business. Ineach of the assays, a homologous pair of specific binding pair ("sbp")members, usually an immunological pair, involving a ligand and areceptor (antiligand) is involved, wherein one of the sbp members islabeled with a label which provides a detectible signal. The immunoassaymethodology results in a distribution of the signal label between signallabel bound in a complex of the sbp members and unbound signal label.The differentiation between bound and unbound signal label can be as aresult of physical separation of bound from unbound signal label ormodulation of the detectible signal between bound and unbound signallabel.

For the most part, immunoassays have been directed to quantitativedetermination of a wide variety of compounds of interest in clinicallaboratories requiring relatively sophisticated equipment and carefultechnique. Immunoassays have found less extensive commercial applicationwhere semi-quantitative or qualitative results would be acceptable andthe determination would involve non-laboratory personnel, such as in ahome or a medical practitioner's office. Even in the clinicallaboratory, simple and rapid screening tests employing inexperiencedpersonnel could serve to provide substantial economies.

In developing an immunoassay, there are many considerations. Oneconsideration is to provide substantial differentiation between theobserved signal resulting from signal label when bound as compared tounbound. Another consideration is to minimize interference fromendogenous materials in the sample suspected of containing the compoundof interest. A further consideration is the ease with which the observedsignal can be detected and serve to differentiate between concentrationsin the concentration range of interest. Other factors include the easeof preparation of the reagents, the accuracy with which samples andreagent solutions must be prepared and measured, the storage stabilityof the reagents, the number of steps required in the protocol, and theproficiency and accuracy with which each of the steps must be performed.Therefore, in developing an assay which can have application withuntrained personnel, such as assays to be performed in the home, inforensic medicine, by medical practitioners, or the like, the observedresult should be minimally affected by variations in the manner in whichthe protocol is carried out or provide for simple techniques forperforming the various steps.

2. Description of the Prior Art

A test device for determining a characteristic of a sample, particularlyfor determining substances in fluid samples, is disclosed in U.S. Pat.No. 4,094,647. A thin layer chromatography device and method of making achromatography test is disclosed in U.S. Pat. No. 4,384,958. Animmunoassay wherein labeled antibody is displaced from immobilizedanalyte analog is described in U.S. Pat. No. 4,434,236. A device andmethod for detecting myoglobin is disclosed in U.S. Pat. No. 4,189,304.Test strips for analyzing substances dissolved in liquids are describedin U.S. Pat. No. 4,438,067. A multi-layered test device for determiningthe presence of a liquid sample component and the method of using such adevice, are described in U.S. Pat. No. 4,160,008. A method for measuringantigen by labeled antigen using insoluble antibody is disclosed inJapanese patent application Laid-Open No. 5925/73--Jan. 25, 1973.

A concentrating zone method in heterogeneous immounoassays is disclosedin U.S. Pat. No. 4,366,241. U.S. Pat. No. 4,168,146 describes animmunoassay test strip. U.S. Pat. Nos. 3,990,850 and 4,055,394 describediagnostic test cards. An automated method for quantitative analysis ofbiological fluids is described in U.S. Pat. No. 4,327,073. A chromogenicsupport immounoassay is disclosed in International Application No.PCT/US83/01887.

A wide variety of patents and patent applications provide an extensiveliterature of different techniques for producing detectible signals inimmunoassays. The following list is merely illustrative of some of thesetechniques which can find application in this invention. The followingis a list of U.S. Pat. Nos. and patent applications and a generalstatement of the type of label involved:

U.S. Pat. Nos. 3,646,346, Radioactive Label; 3,654,090, 3,791,932 and3,817,838, Enzyme Labels; 3,996,345, Fluorescer-Quencher Labels;4,062,733, Radioactive Label; 4,067,959, Fluorescer or Enzyme Label;4,104,029, Chemiluminescent Label; and 4,160,645, Non-Enzymatic CatalystLabel. See U.S. Pat. Nos. 3,966,879 for an electrophoretic techniqueemploying an antibody zone and 4,120,945 for an RIA where labeledanalyte is initially bound to a soild support through antibody. U.S.Pat. No. 4,233,402 employs enzyme pair labels; U.S. Pat. No. 4,720,450,chemically induced fluorescent labels; and U.S. Pat. No. 4,287,300,enzyme anionic charge labels.

SUMMARY OF THE INVENTION

The methods and devices of the present invention are useful fordetermining the presence of an analyte in a sample suspected ofcontaining the analyte. The device is a strip of bibulous materialcapable of being traversed by a test solution through capillarymigration. The test solution is comprised of the sample and a firstmember of a specific pair ("sbp member") capable of binding the analyte.The strip contains, integral therewith, a second sbp member forconcentrating and non-diffusively binding the first sbp member at asmall situs on the strip separate from an end portion of the stripprovided for contacting with the test solution. Generally, the secondsbp member binds to a complex formed from the binding of the analyte tothe first sbp member. A detectible signal is produced by means of asignal producing system. The signal is produced in relation to thepresence of analyte in the test solution. In one embodiment an analog ofthe analyte is non-diffusively bound to the strip at least between thesitus and the portion of the strip that contacts the test solution.

In the method an end portion of the strip separated from the situs iscontacted with the test solution, which traverses the bibulous materialby means of capillary action. The strip is contacted with a developersolution containing members of the signal producing system and then withany remaining members of the signal producing system that were notincluded in the test solution or the developer solution, or presentinitially on the strip. At least a portion of the test solution contactsthe situs prior to contact of the developer solution with the situs. Thesignal detectible at the situs is then compared with the signaldetectible at a portion of the strip other than at the situs todetermine the presence of the analyte in the test solution.

In a particular embodiment of the present invention the signal producedat the small situs has a sharp-edged distinctive pattern that provides asharp contrast to the signal produced at portions of the strip otherthan at the situs when analyte is present in the test solution.

In another particular embodiment of the present invention, the secondsbp member is non-diffusively bound to a small situs on the stripthrough the intermediacy of particles non-diffusively bound to the smallsitus. Where the second sbp member is able to bind the first sbp memberwhen the first sbp member is not bound to the analyte an analog of theanalyte capable of binding the first sbp member is non-diffusively boundto the strip between the situs and the end portion.

The method and device of the present invention have particularapplicability to the determination of a plurality of analytes in a testsolution. The presence or absence of one or more analytes in the testsolution can be readily determined on a single strip. In addition, themethod of the invention provides for the detection of analytes, such asdrugs, without the need for reference materials or instrumentation.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

As mentioned above, the present invention is directed to methods anddevices for determining the presence of an analyte in a sample suspectedof containing the analyte. A test solution is formed by combining in anaqueous medium the sample and a first sbp member capable of binding theanalyte. The end portion of a strip of bibulous material capable ofbeing traversed by this test solution by means of capillary migration iscontacted with the test solution. The strip contains, integraltherewith, a second sbp member capable of binding the complex formedfrom the analyte and the first sbp member. The second sbp member isnon-diffusively bound at a small situs on the strip separate from theend portion. The test solution is allowed to traverse at least a portionof the strip. Next, the strip is contacted with a developer solutioncontaining members of the signal producing system. In the method atleast a portion of the test solution contacts the situs prior to contactof the situs with the developer solution. The strip is then contacted,where necessary, with any remaining members of the signal producingsystem that were not included in the test solution or the developersolution or present on the strip. The detectible signal at the situs isthen compared with the signal detectible at a portion of the strip otherthan at the situs. A signal is produced at the situs in relation to thepresence of analyte in the test solution.

The second sbp member provides a means for concentrating andnon-diffusively binding the first sbp member to the strip at the situs.The first sbp member is part of a signal producing system which providesa detectible signal at the situs in relation to the amount of analyte inthe sample. The surface area of the situs is substantially less thanthat of the strip. The second sbp member has the characteristic ofbinding a complex of the first sbp member by binding directly to theanalyte, by binding directly to the first sbp member, including bindingto the signal generating label, or by binding at a site that is presentonly in the complex. Where the second sbp member is able to bind thefirst sbp member directly and is therefore able to bind uncomplexedfirst sbp member, an analyte analog capable of binding uncomplexed firstsbp member is bound to the strip. The analyte analog normally isnon-diffusively bound to the strip at least between the situs and theend portion.

The means for producing a detectible signal is usually a signalproducing system having one component conjugated to an sbp member toprovide a label-sbp member conjugate. The amount of label producing thedetectible signal is related to the amount of analyte in the testsolution. The signal producing system comprises the label-sbp memberconjugate and all other regents required to produce a detectible signalat the situs in relation to the presence or amount of analyte in thesample.

In a particular embodiment of the present invention the second sbpmember is conjugated to particles, which particles are non-diffusivelybound to the strip at the situs.

The small situs can be a band running transverse to the direction oftraversal of the test solution along the strip. The signal produced atthe small situs can have a sharp-edged distinctive pattern that providesa sharp contrast to signal produced at a portion of the strip other thanthe situs. Usually, the signal generated at the small situs is comparedwith adjacent areas on the strip.

The present invention is particularly applicable to the determination ofthe presence of a plurality of analytes in a test solution.

Before proceeding further with the description of the specificembodiments of the present invention, a number of terms will be defined.

Analyte--the compound or composition to be measured, which is a memberof a specific binding pair and may be a ligand, which is mono- orpoly-valent, usually antigenic or haptenic, a single or plurality ofcompounds which share at least one common epitopic or deteminant site,or a receptor.

The polyvalent ligand analytes will normally be poly(amino acids), i.e.,polypeptides and proteins, polysaccharides, nucleic acids, andcombinations thereof. Such combinations include bacteria, viruses,chromosomes, genes, mitochondria, nuclei, cell membranes and the like.

The precise nature of the analytes together with numerous examplesthereof are disclosed in U.S. Pat. No. 4,299,916 to Litman, et al.,particularly columns 16 to 23, the disclosure of which is incorporatedherein by reference.

Member of a specific binding pair ("sbp member")--one of two differentmolecules, having an area on the surface or in a cavity whichspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of the other molecule. Themembers of the specific binding pair are referred to as ligand andreceptor (antiligand). These will usually be members of an immunologicalpair, although other specific binding pairs such as biotin-avidin,hormones-hormone receptors, nucleic acid duplexes and the like are notimmunological pairs.

Ligand--any organic compound for which a receptor naturally exists orcan be prepared.

Receptor ("antiligand")--any compound or composition capable ofrecognizing a particular spatial and polar organization of a molecule,e.g., epitopic or determinant site. Illustrative receptors includenaturally occurring receptors, e.g., thyroxine binding globulin,antibodies, enzymes, Fab fragments, lectins, nucleic acids, protein A,complement component Clq, and the like.

Ligand analog or analyte analog--a modified ligand or ligand surrogateor modified analyte or analog surrogate which can compete with theanalogous ligand or analyte for a receptor, the modification providingmeans to join a ligand analog or analyte analog to another molecule. Theligand analog or analyte analog will usually differ from the ligand oranalyte by more than replacement of a hydrogen with a bond which linksthe ligand analog or analyte analog to a hub or label, but need not. Theterm ligand surrogate or analyte surrogate refers to a compound havingthe capability of binding the first sbp member. Thus, the ligandsurrogate or analyte surrogate may bind to the first sbp member in amanner similar to the ligand or analyte. On the other hand, thesurrogate could be, for example, an antibody directed against theidiotype of an antibody to the ligand or analyte.

Bibulous material--a porous material having pores of at least 0.1μ,preferably at least 1.0μ, which is susceptible to traversal by anaqueous medium in response to capillary force. Such materials aregenerally hydrophilic or are capable of being rendered hydrophilic andinclude inorganic powders such as silica, magnesium sulfate, andalumina; natural polymeric materials, particularly cellulosic materialsand materials derived from cellulose, such as fiber containing papers,e.g., filter paper, chromatographic paper, etc.; synthetic or modifiednaturally occurring polymers, such as nitrocellulose, cellulose acetate,poly(vinyl chloride), polyacrylamide, cross linked dextran, agarose,polyacrylate, etc.; either used by themselves or in conjunction withother materials; ceramic materials; and the like. The bibulous materialcan be attached to a support. On the other hand, the bibulous materialmay provide its own support. The bibulous material may be polyfunctionalor be capable of being polyfunctionalized to permit covalent bonding ofsbp members as well as to permit bonding of other compounds which form apart of the signal producing system.

Binding of sbp members to the bibulous material may be accomplished bywell-known techniques, commonly available in the literature. See, forexample, "Immobilized Enzymes," Ichiro Chibata, Halsted Press, New York(1978) and Cuatrecasas, J. Bio. Chem., 245: 3059 (1970).

The bibulous material can be a single structure such as a sheet cut intostrips or it can be particulate material bound to a support or solidsurface such as found, for example, in thin-layer chromatography.

The support for the bibulous material where a support is desired ornecessary will normally be water insoluble, non-porous, and rigid andusually will be of the same length and width as the bibulous strip butmay be larger or smaller. A wide variety of organic and inorganicmaterials, both natural and synthetic, and combinations thereof, may beemployed provided only that the support does not interfere with thecapillary action of the strip, or non-specifically bind assaycomponents, or interfere with the signal producing system. Illustrativepolymers include polyethylene, polypropylene, poly(4-methylbutene),polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon,poly(vinyl butyrate), glass, ceramics, metals, and the like.

Labeled-sbp member--a label, for example, a catalyst, usually an enzyme,conjugated to an sbp member, which is a member of the signal producingsystem. The sbp member can bind directly to the analyte or can bindindirectly to the analyte by binding to an sbp member complementary tothe analyte.

Label--A label may be any molecule conjugated to another molecule or tothe bibulous support and, where two molecules are involved, isarbitrarily chosen as to which molecule is the label. In the subjectinvention, the labels will be a member of the signal producing systemthat is conjugated to an sbp member. The label may be isotopic ornonisotopic, preferably nonisotopic. However, an iostopic label can bepreferred for achieving high sensitivity when using radio-autographicdetections with photographic film.

Signal Producing System--The signal producing system may have one ormore components, at least one component being the label conjugated to ansbp member. The signal producing system includes all of the reagentsrequired to produce a measurable signal. When the first sbp member isnot conjugated to a label, the label is normally bound to an sbp membercomplementary to the first sbp member and is usually included as part ofthe developer. Other components of the developer include substrates,coenzymes, enhancers, second enzymes, activators, cofactors, inhibitors,scavengers, metal ions, specific binding substances required for bindingof signal generating substances, and the like. The components of thesignal producing system may be bound to the strip such as coenzymes,substances that react with enzymic products, other enzymes andcatalysts, and the like. The signal producing system provides a signaldetectable by external means, normally by measurement of electromagneticradiation, desirably by visual examination. For the most part, thesignal producing system includes a chromophoric substrate and enzyme,where chromophoric substrates are enzymatically converted to dyes whichabsorb light in the ultraviolet or visible region, phosphors orfluorescers.

The signal-producing system can include at least one catalyst, usuallyat least one enzyme, and at least one substrate and may include two ormore catalysts and a plurality of substrates, and may include acombination of enzymes, where the substrate of one enzyme is the productof the other enzyme. The operation of the signal producing system is toproduce a product which provides a detectable signal at the small situs,related to the amount of catalyst bound to the situs, as a result of sbpmember complex formation of the labeled sbp member.

The signal producing system provides for the production of a compound,which is normally the signal generating compound, but in some instancesmay react with another compound bound to the surface with theproduction, enhancement or destruction of the signal generatingcompound. While both enzymatic and non-enzymatic catalysts may beemployed, usually there will be at least one enzyme catalyst employed inthe signal producing system. In the event of there being only onecatalyst, this catalyst will usually be conjugated to an sbp member forbinding to the situs through sbp member complex formation. In additionto the catalyst, there must be a substrate which undergoes atransformation which results in a change in a detectable signal at themeasurement surface. For the most part, the product resulting from thetransformation catalyzed by the labeled sbp member will be the signalgenerating compound.

Two catalysts may be employed, either a combination of an enzyme and anon-enzyme catalyst or two enzymes, where the two catalysts are relatedin that the product of one is the substrate of the other. In thissystem, there need be only one substrate which can undergo successivechanges catalyzed by the catalysts, which results in the compoundinvolved with production of a detectable signal. For the most part,however, there will normally be a substrate for the first enzyme in theseries and a second compound, which serves as a precursor to thecompound involved in the production of the signal, normally providingthe compound which produces the signal. Thus, the product of the firstenzyme may react with the precursor to the signal producing compound toprovide the signal generating compound.

For the most part, the involved reactions will be hydrolysis or redoxreactions. In the case of hydrolysis, a derivatized dye precursor thathas an enzymatically labile bond and an enzyme that catalyzes itsconversion to an insoluble dye product, is illustrative of this type ofsystem. In redox reactions, a first enzyme would produce an essentialoxidizing substrate required for the second enzyme, where the secondenzyme catalyzes the reaction between the oxidizing substrate and a dyeprecursor.

Where two enzymes are used, the first enzymatic reaction may involvehydrolytic cleavage or a redox reaction of the substrate to provide aproduct which is the substrate of another enzyme. The first situationmay be illustrated by glucose-6-phosphate being catalytically hydrolyzedby alkaline phosphatase to glucose, where glucose is a substrate forglucose oixidase. The second situation may be illustrated by glucosebeing oxidized by glucose oxidase to provide which would enzymaticallyreact with a leuco dye to produce a signal generator.

Coupled catalysts can also involve an enzyme with a non-enzymaticcatalyst. The enzyme can produce a reactant which undergoes a reactioncatalyzed by the non-enzymatic catalyst or the non-enzymatic catalystmay produce a substrate (includes coenzymes) for the enzyme. A widevariety of non-enzymatic catalysts which may be employed are found inU.S. Pat. No. 4,160,645, issued July 10, 1979, the appropriate portionsof which are incorporated herein by reference.

Various combinations of enzymes may be employed to provide a signalgenerating compound. Particularly, combinations of hydrolases may beemployed to produce an insoluble signal generator. Alternatively,combinations of hydrolases and oxidoreductases can provide the signalgenerating compound. Also, combinations of oxidoreductases may be usedto produce an insoluble signal generating compound.

For combinations of enzymes one enzyme can be non-diffusively bound tothe strip, while the other enzyme is conjugated to a sbp member.Additionally, one or more other members of the signal producing systemcan be bound to the strip depending on the particular signal producingsystem chosen or the particular protocol followed.

In order to have a detectable signal, it is desirable to provide meansfor amplifying the signal produced by the presence of the label bound atthe situs. Therefore, it will usually be preferable for the label to acatalyst or luminescent compound or radioisotope, most preferably acatalyst. Preferably catalysts are enzymes and coenzymes which canproduce a muliplicity of signal generating molecules from a singlelabel.

An enzyme or coenzyme is employed which provides the desiredamplification by producing a product, which absorbs light, e.g., a dye,or emits light upon irradiation, e.g., a fluorescer. Alternatively, thecatalytic reaction can lead to direct light emission, e.g.,chemiluminescence. A large number of enzymes and coenzymes for providingsuch products are indicated in U.S. Pat. No. 4,275,149 bridging columns19 to 23, and U.S. Pat. No. 4,318,980, columns 10 to 14, whichdisclosures are incorporated herein by reference.

Of particular interest is the use of a combination of enzymes, where theenzymes are related by the product of one enzyme being the substrate ofthe other enzyme. In this manner, stable precursors to labile substratescan be provided and the substrate for a second enzyme can be stored incombination with a first enzyme without a reaction being prematurelyinitiated.

A number of enzyme combinations are set forth in U.S. Pat. No.4,275,149, bridging columns 23 to 28, which combinations can find use inthe subject invention. This disclosure is incorporated herein byreference.

Of particular interest are enzymes which involve the production ofhydrogen peroxide and the use of the hydrogen peroxide to oxidize a dyeprecursor to a dye. Particular combinations include saccharide oxidases,e.g., glucose and galactose oxidase, or heterocyclic oxidases, such asuricase and xanthine oxidase, coupled with an enzyme which employs thehydrogen peroxide to oxidize a dye precursor, that is, a peroxidase suchas horse radish peroxidase, lactoperoxidase, or microperoxidase.Additional enzyme combinations may be found in the subject matterincorporated by reference. When a single enzyme is used as a label,other enzymes may find use such as hydrolases, transferases, andoxidoreductases, preferably hydrolases such as alkaline phosphatase andβ-galactosidase. Alternatively luciferase may be used such as fireflyluciferase and bacterial luciferase.

Illustrative coenzymes which find use include NAD[H]; NADP[H], pyridoxalphosphate; FAD[H]; FMN[H], etc., usually coenzymes involving cyclingreactions, see particularly U.S. Pat. No. 4,318,980.

The product of the enzyme reaction will usually be a dye or fluorescer.A large number of illustrative fluorescers are indicated in U.S. Pat.No. 4,275,149, columns 30 and 31, which disclosure is incorporatedherein by reference.

Ancillary Materials--Various ancillary materials will frequently beemployed in the assay in accordance with the present invention. Forexample, buffers will normally be present in the assay medium, as wellas stabilizers. Frequently, in addition to these additives, additionalproteins may be included, such as albumins, or surfactants, particularlynon-ionic surfactants, binding enhances, e.g., polyalkylene glycols, orthe like.

Small situs--an area on the strip of bibulous material which has asurface area substantially less than the surface area of the strip. Thesitus may be a dot, line, curve, band, pattern formed from dots, lines,curves, bands, or combinations thereof, or the like. Generally, thedirection of traversal of the strip by the test solution will betransverse to the situs. Preferably, the signal produced at the situshas a sharp-edged distinctive pattern that provides a sharp contrast tosignal produced at portions of the strip other than the situs. Forexample, the situs can be a printed display of an abbreviated name ornames of the analyte or analytes in the test solution, of a plus sign,or of the like. The situs is separated from the end portion of the stripcontacted with the test solution in accordance with the concentratingprinciple of the present invention. The situs should contact a majorportion of the solution flowing through the strip for efficientconcentration.

In the method of the invention, a first sbp member capable of binding tothe analyte is combined with a sample suspected of containing theanalyte to provide an aqueous test solution. A second sbp member capableof binding the complex formed upon binding of the analyte to the firstsbp member is non-diffusively bound to the bibulous strip at the smallsitus. One end of the strip is contacted with the test solution, whichwill traverse the strip through capillary action. The amount of thefirst sbp member that becomes bound to the situs through binding to thesecond sbp member is related to the amount of analyte in the sample. Thesignal producing system provides a detectible at the situs only when thefirst sbp member is bound, so that the presence of the analyte may bedetermined by comparing the signal detectible at the situs with thesignal detectible at a portion of the strip other than at the situs,usually a portion of the strip ajacent to the situs. The first sbpmember binds specifically to the analyte. The second sbp member isnon-diffusively bound at the situs and is able to bind the first sbpmember. Binding may occur directly to a binding site on the first sbpmember, or indirectly to a binding site on the analyte which is bound tothe first sbp member. Binding may also occur at a site characteristic ofthe complex of the analyte and first sbp member which site is notpresent in either component alone.

Where binding of the first sbp member to the second sbp member occursdirectly, it is necessary to provide for removal of free first sbpmember by providing an analog of the analyte non-diffusively bound tothe strip at least between the situs and the end portion. A second sbpmember will normally be chosen that provides for direct binding to thefirst sbp member when the analyte has a single binding site, e.g., adrug, or when only one sbp member complementary to the analyte isavailable. Generally, the amount of analog analyte bound to the stripshould be sufficient to bind all of the first sbp member when no analyteis present in the test solution. Usually, such analog will be present inan excess amount.

The movement of the test solution along the strip is due to capillarity.This capillary movement along the strip causes the test solution to becarried through the situs. Preferably after the strip has been contactedwith the test solution, a developer solution provides for continuationof the capillary migration through the situs. In this instance thedeveloper solution is contacted with the end portion of the strip whichwas contacted with the test solution. Alternatively, to contact thestrip with the developer solution, the situs can be immersed in thedeveloper solution after the end portion of the strip has been contactedwith the test solution. In any event one desires to provideconcentration of the first sbp member at the situs prior to contact ofthe situs with the developer.

The solvent will normally be an aqueous medium, which may be up to about40 weight percent of other polar solvents, particularly oxygenatedsolvents of from 1 to 6, more usually of from 1 to 4 carbon atoms,including alcohols, ethers and the like. Usually, the cosolvents will bepresent in less than about 20 weight percent.

The pH for the medium will usually be in the range of 4-11, more usually5-10, and preferably in the range of about 6-9. The pH is chosen tomaintain a significant level of binding affinity of the sbp members andoptimal generation of signal by the signal producing system. Variousbuffers may be used to achieve the desired pH and maintain the pH duringthe assay. Illustrative buffers include borate, phosphate, carbonate,tris, barbital and the like. The particular buffer employed is notcritical, but in individual assays, one buffer may be preferred overanother.

Desirably, from about 0.05 to 0.5 wt.% of a non-ionic detergent isincluded with the sample. Various polyoxyalkylene compounds may beemployed of from about 200 to 20,000 daltons.

Moderate, and desirably substantially constant, temperatures arenormally employed for carrying out the assay. The temperatures for theassay and production of a detectable signal will generally be in therange of about 4°-50° C., more usually in the range of about 10°-40° C.,and frequently will be ambient temperatures, that is, about 15°-25° C.

The concentration, in the liquid sample, of analyte which may be assayedwill generally vary from about 10⁻⁴ to about 10⁻¹⁵ M, more usually fromabout 10³¹ 6 to 10⁻¹⁴ M. Considerations, such as the concentration ofthe analyte of interst and the protocol will normally determine theconcentration of the other reagents.

While the concentrations of many of the various reagents in the sampleand reagent solutions will generally be determined by the concentrationrange of interest of the analyte, the final concentration of each of thereagents will normally be determined empirically to optimize thesensitivity of the assay over the range of interest. With certainprotocols, individual reagents may be used in substantial excess withoutdetrimentally affecting the sensitivity of the assay.

The size of the strip is dependent on several considerations. Whencapillary flow is predominantly upward, the length and thickness of thestrip control, the amount of solution that can pass through the situs.The transfer of a large volume of first solution requires that the fluidcapacity of the strip above the situs be sufficient to accommodate thedesired volume. If the strip is used to provide a predominantly downwardflow so as to syphon the test solution this volume requirement is notneeded. Moreover, if an absorbent material is provided to contact theend of the strip not used to contact the test solution the volumerequirement is also eliminated. In general, it is desirable to transferas large a volume as possible through the situs so as to provide thegreatest assay sensitivity. However, other considerations such as timeand the availability of the sample will limit this requirement. Ingeneral, for upward flow strips the fluid retention volume about thesitus will be usually greater than 20 μL, preferably at least 50-200 μL.For downward flow strips retention volumes as low as 2-20 μL can be usedbut volumes of 20-200 μL are preferable.

Thickness of the strips will usually be no greater than 20% of thewidth, preferably 1 to 10%, more preferably 2 to 5%.

To permit conservation of reagents and provide for samples of limitedsize, the width of the strip will generally be relatively narrow,usually less than 20 mm, preferably less than 10 mm. Generally, thewidth of the strip will not be less than about 1.0 mm and will usuallyrange from about 2 mm to 12 mm, preferably from about 4 mm to 8 mm.

The length of the strip will depend on the concentration of the analyteand practical considerations such as ease of handling and the number ofsituses on the strip and will be about 2 cm to 40 cm, usually about 4 cmto 25 cm, preferably about 6 to 20 cm but may be of any practicallength. The structure of the strip can be varied widely and includesfine, medium fine, medium, medium coarse and coarse. In general, smallerpore size and finer material will provide slow capillary flow andefficient capture of binding sbp members on the strip. Courser, moreporous materials provide faster flow, but the efficiency of capture isreduced. Selection of the porosity of the material depends on the rateof binding of the sbp members for a given assay.

The position of the small situs, or small situses where a plurality ofanalytes are being determined, should be governed by the basic principleinvolved in the present invention. One desires to pass by capillarity asufficient amount of the test solution through the situs to concentratea sufficient amount of an analyte at the situs to produce a signal thatis detectible over the background. Thus, it is desirable to position thesitus close to the end of the strip which is to contact the testsolution but not so close as to come into contact with the bulk solutionor the menicus. Desirably, the situs should be at least 5 mm, preferablyat least 8 mm, from such end of the strip. It may be positioned anygreater distance away from the end provided the test solution can passthrough the situs by capillary action. Preferably, the situs will not begreater than half the length of the strip from such end. In this way,the situs is "separated" from such end portion. Where several situsesare used, the situses can be grouped close together or apart but mustnot be so close as to compromise resolution of the signal. Consequently,such situses usually should be spaced not less than 1 mm apart,preferably at least 3 mm apart.

Other reagents which are members of the signal producing system can varywidely in concentration depending upon the particular protocol and theirrole in signal production. Usually the first sbp member will not exceed10⁴ times the maximum concentration of interest of the analyte when theanalyte has multiple binding sites and will not exceed 10³ times themaximum concentration of interest when a monovalent analyte is used.Normally, the first sbp member will not be less than about 0.5 times theminimum concentration of interest. When the label is not bound directlyto the first sbp member, the reagent to which it is bound must bind tothe first sbp member and will be present in at least an amountequivalent to the lowest concentration of interest of the analyte.

In carrying out the assay, the protocol will normally involve combiningthe sample suspected of containing the analyte with the first sbp memberto form the aqueous test solution. The sample may be derived from a widevariety of sources, such as physiologic fluids, illustrated by saliva,blood, serum, plasma, urine, ocular lens fluid, spinal fluid, etc.,chemical processing streams, food waste water, etc.

The end portion of the strip, usually, the end closest to the situ, iscontacted with the test solution, usually by immersion of the endportion into the test solution. Wetting of the strip by capillary actionusually is allowed to continue at least until the situs is wet.Preferably, at least half the strip is wet with the test solution. Whendownward syphoning flow is used, usually the entire strip will be wetand excess test solution can be allowed to syphon through the strip.

For the most part, relatively short times are involved for the testsolution to traverse the strip. Usually, the traverse of the testsolution over the strip will take at least 30 sec and not more than 1hour, more usually from about 1 min to 30 min. The development of thesignal will generally range from 30 sec to 30 min, more usually fromabout 30 sec. to 5 min.

After the liquid has traversed at least a portion of the strip, thestrip is contacted with a developer solution having members of thesignal producing system. This may be accomplished by immersion of thestrip into the developer solution, but preferably only the end of thestrip previously in contact with the test solution is contacted with thedeveloper solution. Where the test solution contains unlabeled first sbpmember, the developer solution will have a labeled sbp member that canbind to the complex formed between the first sbp member and the analyte.Upon contact of the end portion of the strip with the developersolution, the solution traverses the strip by capillary action at leastto the small situs and preferably until the entire strip is wet.

When an enzyme is used as a label, the substrate will normally be insubstantial excess, so as not to be rate limiting (greater concentrationthan Km). The developer solution will usually be appropriately bufferedfor the enzyme system.

After contacting the strip with the developer solution, the strip iscontacted with any remaining members of the signal producing system notpresent in the developer or test solutions or present on the strip. Asufficient time is allowed to elapse prior to measuring the signal toproduce an amount of the signal producing compound required to definethe region of the situs in which the analyte is bound. Once thedetectable signal has been produced, the presence or absence of theanalyte or analytes in the sample is known.

The ligand analytes are characterized by having single binding sites(monovalent) or multiple binding sites (polyvalent), while the reactoranalytes may also have a single or plurality of binding sites. Thepolyvalent analytes will normally be poly(amino acids), i.e.,polypeptides and proteins, polysaccharides, nucleic acids, andcombinations thereof. Such combinations or assemblages include bacteria,viruses, chromosomes, genes, mitochondria, nuclei, cell membranes andthe like.

For the most part, the polyvalent ligand analytes will have a molecularweight of at least about 5,000, more usually at least about 10,000. Inthe poly(amino acid) category, the poly(amino acids) of interest willgenerally be from about 5,000 to 5,000,000 molecular weight, moreusually from about 20,000 to 1,000,000 molecular weight, and amonghormones of interest, about 5,000 to 60,000 molecular weight.

An extensive listing of useful ligands may be found in U.S. Pat. No.4,275,149, the disclosure bridging columns 12 to 17, which disclosure isincorporated herein by reference.

The monovalent ligand analytes will generally be from about 100 to 2,000molecular weight, more usually from about 125 to 1,000 molecular weight.The analytes of interest include drugs, hormones, metabolites,pesticides, pollutants, and the like.

A large number of analytes of interest are listed in U.S. Pat. No.4,275,149, columns 17 and 18, which disclosure is incorporated herein byreference.

For receptor analytes, the molecular weights will generally range fromabout 10⁴ to 2×10⁸, more usually from about 3×10⁴ to 2×10⁶. Forimmunoglobulins, e.g., IgA, IgD, IgE, IgG and IgM, the molecular weightswill generally vary from about 160,000 to about 10⁶. Enzymes willnormally vary from about 10,000 to 600,000 daltons. Natural receptorsvary widely, being generally at least about 25,000 molecular weight andmay be 10⁶ and higher, including such materials as avidin, thyroxinebinding globulin, thyroxine binding prealbumin, transcortin, membranesurface proteins, etc.

Where a lignad is conjugated to another molecule or support, frequentlythe ligand will be modified to provide for a particular functional groupat a particular site. This modification produces a product referred toas a ligand analog. U.S. Pat. No. 4,275,149 also has an extensivedescription of ligand analogs, bridging columns 18 and 19, whichdescription is incorporated herein by reference.

The strip can be coated with a wide variety of materials to provide forenhanced properties. Coatings may include protein coatings,polysaccharide coatings, synthetic polymers, sugars or the like, whichare used particularly to enhance the stability of the materialsconjugated to the support. These compounds can also be used for improvedbinding of the materials, such as the sbp member or signal producingsystem member bound to the strip.

The strip, or the situs, can be activated with reactive functionalitiesto provide for covalent bonding of the organic materials to beconjugated to the strip such as those described in U.S. Pat. No.4,168,146.

The amount of sbp member which is bound to the strip at the situs willvary depending upon the amount required to bind all of the labeled sbpmember. Generally, the anount of sbp member at the situs will be atleast equivalent to the amount of analyte that flows through the situsand can exceed the amount of analyte by ten thousand fold or more.

The second sbp member, the analyte analog, and, where desired, membersof the signal producing system can be bound to the strip by adsorption,rather than covalent bonding, as long as such binding is non-diffusive.This will involve contacting the bibulous support with a solutioncontaining the materials to be bound to the strip and allowing the stripto dry. In general, this procedure will be useful only where thebibulous support is relatively hydrophobic or has a high surface charge,and subsequent treatment with proteins, detergents, polysaccharides, orother materials capable of blocking non-specific binding sites will berequired.

In a preferred embodiment of the invention the first sbp member can benon-diffusively bound to particles or beads. The particles or beads canthen be applied to the strip at the situs. Generally, the particles willhave means for specifically binding a labeled sbp member or a labelwithout significant non-specific interaction. The nature of the particleor the beads may vary widely, being naturally occurring or synthetic.The materials are commercially available or commercially availablematerials may be modified. Exemplary of such particles or beads arelatex particles made from polystyrene, polyacrylates, polyacrylamide,available as Biogel-p®, or naturally occurring materials such aspolysaccharides, particularly cross-inked polysaccharides, such asagarose, which is available as Sepharose®, dextran, available asSepbadex®, microcrystalline cellulose, starch and the like. Othermaterials include polyacrylamides, polystyrene, polyvinyl alcohol,copolymers of hydroxyethyl methacrylate and methyl methacrylate,silicones, glasses, available as Bioglas®, diatomaceous earth, silica,and the like. The primary requirement is that the materails do notcontribute a signal, usually light absorption, that would cause thesitus to have a different signal than other parts of the strip prior tocontact with the signal producing system.

The particles must be capable of non-diffusivable attachment to an sbpmember where the attachment can be achieved by covalent or non-covalentbinding. When the sbp member is convalently bound, the particles shouldbe polyfunctional or be capable of being polyfunctionalized. A widevariety of functional groups are available or can be incorporated.Functional groups include carboxylic acids, aldehydes, amines, amides,activated ethylenes such as maleimide, hydroxyls, sulfonic acids,mercaptans, and the like. The manner of linking a wide variety ofcompounds to the various particles is well known and is amplyillustrated in the literature. See, for example, Cautrecases, J. Biol.Chem. 245, 3059 (1970).

The length of the linking groups will vary widely depending upon thenature of the compound being linked, the effect of distance between thelabel and the particle on the label's properties, the potential forcross-linking of the labels, and the like.

The particles should not migrate to any significant degree. The size ofthe particles can vary but must be of a size to infiltrate the pores ofthe bibulous material and become imbedded or non-diffusively boundtherein. Thus, the particles are generally slightly larger than theminimum size of the pores of the bibulous material and smaller than themaximum pore size. Usually, the size of the particles will range fromabout 0.1 to 50 microns, more usually from about 0.4 to 10 microns,preferably greater than 0.5μ.

Particles having a non-diffusively bound sbp member may be used tonon-diffusively bind the sbp member to the strip at a small situs withsharply defined edges. Several methods may be employed. Usually asuspension of the particles in a liquid, that frequently is aqueous,will be applied to the strip. Application may be by any standardprinting process including the use of electrostatic and laser propelledjets, and printing probe or type face. In addition, particles could beapplied by template. The shape of the situs would be defiined by a cutpattern through which particles would be absorbed into the bibulousstrip. Alternatively, the suspension can be transferred to the strip byinscribing with a pen or microcapillary tube. Where dry particles areused, they may be applied by directing a jet of a suspension of theparticles in a gas, usually air, at the desired situs. In each case,particularly when printing techniques are not used, it will frequentlybe desirable to provide for reduced pressure on the side of the stripopposite to the side used to apply the particles. Pressure reduction isconveniently provided by placing a sheet of the bibulous material on afilter or porous plate that covers a vacuum chamber. The suspension isthen applied while air is being drawn through the material. Regardlessof the method of application of the particles it is usually preferableto wash the situs free of unbound particles after they have beenapplied.

The liquid used to suspend the particles will usually be aqueous andmust not dissolve the particles or damage or release the bound sbpmember. Thickners and surfactants may be added to limit capillary flowand provide sharply defined edges. Thickners may include polyvinylalcohol, polyprolidone, dextran, glycerol, and the like. Surfactants maybe ionic, usually anionic, or non-ionic.

A variety of embodiments of the present invention will next be describedin some detail. In one embodiment, the analyte in the sample ispolyvalent. A second sbp member which recognizes a determinant site onthe analyte is non-diffusively bound at a small situs on the strip ofbibulous material. The non-diffusive binding may be accomplished by anyof the means described hereinabove. A test solution having a volumeapproximately equal to the fluid capacity of the strip is prepared bycombining the sample which is suspected of containing the analyte and alabeled first sbp member wherein the latter sbp member binds to adeterminant site on the analyte to form a complex. The end portion ofthe bibuluous strip nearest the situs is contacted with the testsolution, which is allowed to traverse the bibulous strip by means ofcapillary action. The test solution moves along the strip and throughthe stius until the entire strip is wet or the test solution isexhausted. The complex of the analyte and labeled first sbp member bindsto the second sbp member at the situs, thereby concentrating the labeledfirst sbp member at the situs. After the test solution is allowed totraverse the bibulous strip, the strip is then contacted with adeveloper solution containing the remaining members of the signalproducing system of which the label is one member. If the analyte ispresent in the test solution, a signal will be produced at the smallsitus. This signal can be distinguished from signal generated adjacentto the situs.

In a variant of the above-described embodiment, the volume of the testsolution is sufficient to permit it to traverse only a portion of thestrip such that the fluid capacity at the dry portion of the strip is atleast as great as the fluid capacity of the portion from the end portionthrough the small situs. The end portion of the strip, which end portionwas previously contacted with the test solution, is next contacted withthe developer solution. The developer solution moves along the stripthrough the small situs by capillarity. In doing so, the developersolution causes the remainder of the test solution to move through thesmall situs. If analyte is present in the test solution, a signal isgenerated.

In another variant of the above-described embodiment the first sbpmember is not labeled. The assay is carried out in the same way but alabeled sbp member complementary to the first sbp member is included inthe developer. When analyte is present, the first sbp member binds tothe situs and thereby causes binding of the labeled sbp member to thesitus. In this embodiment, it is often preferable to exclude somemembers of the signal producing system from the developer and to contactthe strip with a solution containing these excluded, i.e., remaining,members after contact with the developer.

In the above embodiment, a plurality of polyvalent analytes may bedetermined. To this end, several situses separated from the end portionare employed. At one situs a second sbp member which recognizes adeterminant site on a first analyte is non-diffusively bound to thebibulous strip. At another situs another second sbp member whichrecognizes a determinant site on a second analyte is non-diffusivelybound. The situation continues until there is a small situs for each ofthe analytes for which one desires to test. The sample to be analyzed isthen combined in an appropriate aqueous medium with a plurality oflabeled first sbp members to form the test solution. One labeled firstsbp member will contain an sbp member which recognizes a determinantsite on the first analyte other than the determinant site recognized bythe second sbp member on the bibulous strip which binds to the firstanalyte. Another labeled first sbp member will contain an sbp memberwhich recognizes a determinant site on the second analyte other than thedeterminant site recognized by the second sbp member on the bibulousstrip which binds to the second analyte. The number of labeled sbpmembers will correspond to the number of analytes for which one desiresto test. If the analyte or analytes tested for are present in thesample, a complex of each respective analyte with its correspondinglabeled first sbp member is formed. If the analyte is not present, thenno such complex forms. The end portion of the bibulous strip iscontacted with the test solution, which is allowed to traverse thestrip. Any complexes of analyte and labeled sbp member will bind to therespective situses on the strip. If the analyte is not present, nocomplex forms and, therefore, the labeled first sbp member whichcorresponds to that analyte does not become bound at the situs and asignal will not be produced. After the test solution has traversed thebibulous strip, the strip is contacted with the appropriate members ofthe signal producing systems employed. The assay may be designed so thata single developer is employed for all of the analytes for which onedesires to test. If the particular analyte is present in the sample, asignal will be produced at the appropriate situs. In a particularlypreferred variant of this procedure, the first sbp members are notlabeled, and the developer contains a labeled sbp member complementaryto the first sbp members. The strip is contacted with the remainingmembers of the signal producing system following contact with thedeveloper.

In another embodiment of the present invention, the analyte is amonovalent drug. The sample suspected of containing the drug is mixedwith a labeled first sbp member in an appropriate medium to form theaqueous test solution. The labeled first sbp member binds to the drug.The bibulous strip will contain a second sbp member at the small situs,which sbp member recognizes a determinant site on the labeled first sbpmember other than the determinant site involved in the binding of thelabeled sbp member to the drug. For example, the second sbp member mayrecognize a determinant site on the label portion of the labeled firstsbp member or on the sbp member portion. In this particular embodiment,an analyte or drug analog must be bound to the strip in an amount atleast sufficient to bind all of the labeled sbp member when drug isabsent in the sample tested. Usually, this analyte analog is aderivative of the drug tested for and is bound to the strip in asubstantial excess at least between the end portion of the strip and thesitus. Although it is preferred that this drug analog be a derivative ofthe drug, one may use other drug analogs such as, for example, anantibody directed against the idiotype of an antibody to the drug. Whenthe sample and the labeled first sbp member are mixed together to formthe test solution and the drug is present in the sample, a complexbetween the drug and the labeled first sbp member is formed. Thiscomplex of drug and labeled first sbp member moves along the bibulousstrip until it reaches the situs to which it becomes bound due tobinding with a second sbp member specific for the labeled first sbpmember at the situs. If the drug is not present in the sample, then nocomplex is formed between the drug and the labeled first sbp member.When the test solution is contacted with the end portion of the bibulousstrip, the labeled first sbp member that is not complexed with the drugbinds to the drug analog which is non-diffusively bound to the strip.Since this drug analog is present in excess quantity, the uncomplexedlabeled sbp member does not reach the small situs. In subsequentdevelopment of the test strip, the presence of drug in the sample willbe indicated by production of a signal at the small situs.

In the latter embodiment for monvalent drugs, one may also assay a testsolution for a plurality of drugs. In this situation the test solutionis formed by mixing together in an appropriate liquid medium the sampleand a number of labeled first sbp members corresponding to the number ofanalytes for which one desires to test. If it is only desired to know ifany one of the drugs is present, the bibulous strip contains a situsidentical to that described above for a single drug. It is necessary,however, to include on the strip drug analogs corresponding to each oneof the drug for which one is testing. If it is necessary to know whichdrugs are present, the strip contains a separate situs for each drug onwhich situs is bound a second sbp member that specifically binds to adeterminant site that is characteristic of the labeled first sbp membercorresponding to that drug. A provision for a characteristic determinantsite is preferably made by attaching a hapten to the labeled first sbpmember and using an antibody to the hapten as the second sbp member.

As a matter of convenience, the present device can be provided in a kitin packaged combination with predetermined amounts of reagents for usein assaying for an analyte. Where an enzyme is used as the label, thereagents will include enzyme labeled first sbp member, substrate for theenzyme or precursors therefor including any additional substrates,enzymes and cofactors and any reaction partner of the enzymic productrequired to provide the detectable chromophore or fluorophore. Inaddition, other additives such as ancillary reagents may be included,for example, stabilizers, buffers, and the like. The relative amounts ofthe various reagents may be varied widely, to provide for concentrationsin solution of the reagents which substantially optimize the sensitivityof the assay. The reagent can be provided as dry powders, usuallylyophilized, including excipients, which on dissolution will provide fora reagent solution having the appropriate concentrations for performingthe assay.

EXAMPLES

The invention is demonstrated further by the following illustrativeeample. Before proceeding with a description of the illustrativeexample, a number of terms will be defined.

PBS: phosphate buffer saline

DMF: dimethyl formamide

BSA: bovine serum albumin

HCG: human chorionic gonadotropin

HRP: horseradish peroxidase

PO₄ : mono- and dibasic phosphate, sodium salt

EDTA: ethylenediaminetetraacetic acid

SAMSA: S-acetyl mercaptosuccinic anhydride

SMCC: succinimidyl 4-(N-maleimido methyl) cyclohexane-1-carboxylate

Phosphate Buffer: 10 mM PO₄, pH=7.40,

PBS: 10 mM PO₄, pH=7.40, 150 mM NaCl,

Buffer A: 10 mM PO₄, pH=7.40, 150 mM NaCl, 0.1% BSA, 0.05% Tween-20(Sigma Chemical Co.),

Conjugate Buffer: 100 mM PO₄, pH=7.40, 150 mM NaCl, 0.1% BSA, 0.05%Tween-20.

Developer Buffer: 10 mM PO₄, pH=7.00, 20 mM NaCl, 0.1% BSA, 0.005%Triton QS-44 (Sigma Chemical Co.), 200 μg/ml 4-chloro-1-Naphthol, 1 mMH₂ O₂.

Carbonate Buffer: 50 mM Na₂ CO₃ pH=9.50.

PO₄ --NaCl-EDTA Buffer: 100 mM Na PO₄ pH=7.50, 100 mM NaCl, 5 mM EDTA.

Column Buffer: 100 mM Na₃ PO₄ pH=7.00, 200 mM NaCl, 0.2% NaN₃.

Preparation of Conjugate of HRP and HCG Antibody Preparation ofHRP-SAMSA

Sixty (60) mg of HRP (Type VI, Sigma Chemical Co.) was dissolved in 2 mlof Carbonate Buffer and dialyzed to remove contaminants.

SAMSA (Sigma Chemical Co.) was made 100 mM in dry DMF. Thirty (30) mg ofHRP was reacted with a twelve-fold molar excess of SAMSA for 1 hr. Theproduct was purified from free SAMSA using a small Sephadex G-25 columnequilibrated in PO₄ --NaCl-EDTA Buffer.

To prepare free --SH groups from the SAMSA, the above product wasincubated with a 1/10 volume of 1M NH₂ OH in PBS with stirring underArgon gas for 1 hr. The resulting product was purified on anotherSephadex G-25 column and used immediately.

Preparation of Antibody-SMCC

Monoclonal antibody to the beta subunit of HCG was prepared according tothe procedure of Kohler et al., Nature (1975) 265: 495-497.

The antibody (4-5 mg/ml) was dialyzed against PO₄ --NaCl-EDTA Buffer toremove contaminants. Following dialysis, 8 mg of antibody was reactedwith a 25-fold molar excess of SMCC (Pierce Chemical Co.) for 2 hr. atroom temperature with stirring. The SMCC was prepared fresh as a 100 mMsolution in dry DMF.

The product was purified on a small Sephadex G-25 column and usedimmediately. The column was equilibrated with PO₄ --NaCl-EDTA Buffer.

Conjugation of HRP-SAMSA and Antibody-SMCC

The antibody-SMCC and a twelve-fold molar excess of HRP-SAMSA werecombined and allowed to react at room temperature for 4 hr. withstirring. The reaction was quenched with 1 mM β-mercaptoethanol followed15 minutes later by 2 mM N-ethylmaleimide.

Purification

The above conjugate was separated from free antibody and HRP by sizeexclusion chromatography on a Sephacryl S-300 column equilibrated withColumn Buffer.

Preparation of Antibody-bound Beads

Polybead-carboxylate Microspheres, 3.92 μm diameter 2.5% suspension,from Polysciences, catalog number 9850, were employed. Four hundred(400) μl (10 mgs) of beads were washed with Phosphate Buffer and weresuspended in 5 ml of a 2 mg/ml solution of Phosphate Buffer of amonoclonal antibody prepared in accordance with conventional techniques(Kohler, et al. supra.). This monoclonal antibody recognizes only thealpha subunit of HCG.

The beads were incubated at room temperature overnight in the antibodysolution with stirring to keep the beads suspended. The beads were thencentrifuged, the antibody solution was decanted, and the beads werewashed with 5 ml of each of Phosphate Buffer, PBS, and Buffer A.

After the wash with buffer A, the beads were suspended in 5 ml of PBS+10mg/ml BSA (to block any non-specific binding) and mixed for 1 hour atroom temperature.

Subsequently, the beads were centrifuged and then resuspended in 400 μlof Buffer A.

Preparation of Test Device

Whatman 31 ET paper which had been coated with a protein (polyclonalanti-theophylline) was used. The paper was cut into strips 6 mm wide and9 or 18 cm long depending on the assay protocol. The paper strips wereplaced on a 4.5 cm diameter glass frit filter holder. Vacuum was appliedand the strips were wetted with distilled water.

Ten (10) μl of the antibody-bound beads suspension prepared as describedabove (containing 250 μg beads) was absorbed into the paper by drawing a5 μl capillary tube filled with the antibody-bound beads across thesurface of the strip two times. The beads were then washed into thepaper with 1 ml of Buffer A. The strips were allowed to dry on thevacuum.

ASSAY

A. One hundred (100) μl of HCG solution (20 ng/ml in Buffer A or inurine) was mixed with 100 μl HRP-HCG antibody conjugate solution (1/1000diluted in Conjugate Buffer). A 2 mm portion of a Test Device (9 cm inlength) was immersed in the above mixture, which was allowed to traversethe Test Device until the medium front reached the top of the TestDevice. The Test Device then was immersed in 9 ml of Developer Bufferfor 10 minutes and then was removed. A thin band of blue-gray colorformed across the Test Device where the antibody-bound beads werelocated.

As a control the above procedure was repeated with the exception that100 μl of Buffer A was employed in place of 100 μl of HCG solution. Nocolor band appeared on the Test Device since HCG was not present in thetest solution.

B. One hundred (100) μl of the HCG solution was mixed with 100 μl ofHRP-HCG antibody conjugate solution. A 2 mm portion of a Test Device (18cm in length) was immersed in the above mixture, which traversed theTest Device until the mixture was exhausted (about 10 min). A 2 mmportion of the same Test Device was further immersed in 200 μl ofDeveloper Buffer, which was allowed to traverse the Test Device forabout twenty more minutes. A thin band of blue-gray color formed acrossthe Test Device where the antibody-bound beads were located.

As a control the above procedure was repeated with the exception that100 μl of Buffer A was employed in place of 100 μl of HCG solution. Nocolor band appeared on the Test Device since HCG was not present in thetest solution.

The present invention provides a number of significant advantages overknown methods. A primary advantage of the present invention is that anumber of analytes can be determined in a single assay on a single testelement. This provides a savings in operator's time and in cost. Thereagents and devices can be manufactured easily and inexpensively whichprovides an additional cost savings. The assay result is determined byreference solely to the assay device and when the signal produced is acolor or fluorescence, the device can be ready without the aid of aninstrument. Therefore, a built-in control is provided. A positive resultis easily distinguished over any background produced on the test deviceas the result of non-specific interactions. Also, the factors producingbackground signal affect the situs and the remaining area of the testdevice in substantially the same way.

Another advantage of the present invention is that cumbersome columntechniques are avoided. The assay device is a bibulous strip that iseasy to manipulate. A further advantage is that analyte is concentratedin a small zone, i.e., at the small situs. In many assay situations, theanalyte is present in very small amounts, making detection difficult.Concentrating the analyte in a small zone enhances the accuracy of thedetermination particularly where the analyte is present in smallamounts.

Another advantage of the present invention is that an excess of one ofthe sbp members, generally the labeled sbp member, can be employed.Using an excess of an sbp member aids in driving the reaction to producethe sbp member complexes.

Although the foregoing invention has been described in some detail byway of illustration and example for the purposes of clarity andunderstanding, it will be obvious that certain changes or modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A method for determining the presence of ananalyte in a sample suspected of containing said analyte, which analyteis a member of a specific binding pair ("sbp member") consisting ofligand and its complementary receptor, which method comprises:(a)contacting, with a test solution containing said sample and a first sbpmember capable of binding to said analyte, a contact portion of bibulousmaterial capable of being traversed by said test solution by capillarymigration,said bibulous material containing a second sbp membernon-diffusively bound to a small situs on said bibulous materialseparated from said contact portion, the surface area of said situsbeing substantially less than that of said bibulous material, saidsecond sbp member having the characteristic of binding said first sbpmember when said first sbp member is bound to said analyte, with theproviso that, where said second sbp member is able to bind said firstsbp member when said first sbp member is not bound to said analyte, ananalyte analog capable of binding said first sbp member isnon-diffusively bound to said bibulous material at least between saidsmall situs and said contact portion, (b) allowing the test solution totraverse at least a portion of said bibulous material by means ofcapillary migration, (c) detecting the presence of the first sbp memberat said situs.
 2. The method of claim 1 wherein said bibulous materialis a strip.
 3. The method of claim 1 wherein said first sbp member is anantibody.
 4. The method of claim 1 wherein particles are non-diffusivelybound to said bibulous material at said situs, said particles beingconjugated to said second sbp member.
 5. The method of claim 1 whereinsaid first sbp member is conjugated to a label.
 6. The method of claim 5wherein said first sbp member is detected at said situs by detectingsaid label.
 7. The method of claim 1 wherein the analyte is a drug andthe analyte analog is a drug analog which is non-diffusively bound tosaid bibulous material.
 8. The method of claim 1 wherein the small sizeis a band transverse to the direction of traversal of said test solutionalong said bibulous material.
 9. The method of claim 1 for determiningthe presence of a plurality of analytes in said test solution, wherein aplurality of corresponding second sbp members are each non-diffusivelybound to a small situs on said bibulous material distant from saidcontacting portion wherein each of said second sbp members isrespectively capable of specifically binding a corresponding first sbpmember and said first sbp members are combined with said sample in saidtest solution.
 10. A method for determining the presence of an analytein a sample suspected of containing said analyte, which comprises(a)contacting, with a test solution containing said sample and antibodiesto said analyte, said antibodies being conjugated to a label, a contactportion of bibulous material capable of being traversed by said testsolution by capillary migration,said bibulous material containingparticles non-diffusively bound to a small situs on said bibulousmaterial separate from said contact portion, the surface area of saidsitus being substantially less than that of said bibulous material, saidparticles having a member of a specific binding pair ("sbp member")non-diffusively bound thereto, which sbp member is capable of binding tosaid antibodies to said analyte when said antibodies are bound to saidanalyte, with the proviso that, where said sbp member is able to bindsaid antibodies to said analyte when said antibodies are not bound tosaid analyte, an analog of said analyte capable of binding saidantibodies to said analyte is non-diffusively bound to said bibulousmaterial between said small situs and said contact portion, (b) allowingthe test solution to traverse at least a portion of said bibulousmaterial by capillary migration, (c) detecting the presence of saidfirst sbp member at the situs.
 11. The method of claim 10 wherein saidbibulous material is a strip.
 12. The method of claim 10 wherein saidantibodies to said analyte are antibodies for human chorionicgonadotropin.
 13. The method of claim 10 wherein said sbp member isantibody for human chorionic gonadotropin.
 14. The method of claim 10wherein said antibodies are labelled with an enzyme.
 15. The method ofclaim 14 wherein a second enzyme is bound to said bibulous material, theenzymes being related in that the product of one enzyme is the substrateof the other.
 16. The method of claim 10 for determining a plurality ofanalytes in said sample, wherein a mixture of specific antibodies toeach analyte is combined with the sample and a plurality ofcorresponding sbp members are each non-diffusively bound to a smallsitus on said bibulous material wherein each of said sbp members iscapable of binding at least one of the complexes formed between aspecific antibody and an analyte.
 17. The method of claim 16 wherein ananalyte analog for each of said analytes is bound to said bibulousmaterial.
 18. The method of claim 10 wherein said small situs is a bandtraverse to the direction of traversal of said sample along saidbibulous material.
 19. The method of claim 10 wherein said analyte is adrug and a drug analog is non-diffusively bound to the bibulousmaterial.